Monoclonal antibodies (mAbs) are an increasingly important class of therapeutic agents. Apart from mAb products composed of the full-size form of IgG, a wide variety of multispecific recombinant antibody formats have been developed, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).
Several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N., Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).
All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., Léger, O., Pathobiology 74 (2007) 3-14). Tandem scFVs are two scFv fragments linked by an extra peptide linker and are also referred to as (scFv)2. By reducing the length of the peptide linker between the variable domains, Diabodies are generated. Adding an extra peptide linker between the two polypeptides yields so called single chain diabodies. US 2007/0274985 relates to antibodies comprising single chain Fab (scFab) fragments. Antibody fragments have both pros and cons as therapeutics compared with full-size monoclonal antibodies: One advantage is that they are smaller and penetrate tissues and tumors more rapidly. In addition, the small size of fragments has been suggested to permit binding to epitopes not accessible to full-sized monoclonal antibodies. On the downside, fragments demonstrate short circulating half-lives in humans, likely due to kidney clearance. The shorter half-life may prevent sufficient accumulation of therapy at the targeted site. Production of antibody fragments is not trivial, as fragments are likely to form aggregates and can be less stable than full-size monoclonal antibodies. In addition, unwanted pairing of noncongnate heavy and light chains results in formation of inactive antigen-binding sites and/or other non-functional undesired side-products, which is a major problem in clinical-scale production and therapeutic application of antibody fragments. In particular Tandem-Fab constructs where two or more Fabs are fused with each other via one connector are not feasible due to the random association of the two light chains resulting in inactive, undesired side products. These drawbacks have now been overcome with the new antibody format of the invention. Provided therein is a new bispecific antibody format that can be easily produced with an increased yield due to a decreased amount of mispaired side-products, which show less aggregation than bispecific antibody fragments known in the art. Using the crossover approach correct LC association can be enforced without the need for the generation of a common light chain. Common light chan approach is not possible for existing antibodies. Further, the new bispecific antibody format is stable and does not aggregate at higher temperatures than existing bispecific formats, which allows better producability and developability. In addition, the new bispecific antibody format has a higher molecular weight compared to many conventional bispecific antibody fragments, thus preventing excessive kidney clearance and leading to an improved half-life in vivo. The new bispecific antibody format is fully functional and has comparable or improved binding and activity as corresponding conventional bispecific antibodies.
The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues undamaged. One approach is to selectively induce an immune response against the tumor, which triggers the attack and subsequent destruction of tumor cells by immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs). CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies. In this regard, bispecific antibodies which are able to bind to a surface antigen on cancer cells and to an activating invariant component of the cell receptor (TCR) complex have become of interest in recent years. The simultaneous binding of the bispecific antibody to both of its targets forces a temporary interaction between cancer cell and T cell, causing activation of cytotoxic T cells and subsequent lysis of the tumor cell.
Several bispecific antibody formats have been developed and their suitability for T cell mediated cancer immunotherapy investigated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promising results in the clinic (reviewed in Nagorsen and Baiuerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker. Further bispecific formats being evaluated for T cell engagement include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent development are the so-called DART (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). The so-called triomabs, which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical trials, represent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).
However, the bispecific antibodies developed for T cell mediated cancer immunotherapy known so far have major drawbacks relating to their efficacy, toxicity and applicability. Small constructs such as, for example, BiTE molecules—while being able to efficiently crosslink effector and target cells—have a very short serum half life requiring them to be administered to patients by continuous infusion. IgG-like formats on the other hand—while having the great benefit of a long half life—suffer from toxicity associated with the native effector functions inherent to IgG molecules. This immunogenic potential constitutes another unfavorable feature of IgG-like bispecific antibodies, for successful therapeutic development. Finally, a major challenge in the general development of bispecific antibodies remains the production of bispecific antibody constructs at a clinically sufficient quantity and purity. The mispairing of antibody heavy and light chains of different specificities upon co-expression, decreases the yield of the correctly assembled construct and results in a number of non-functional side products.
Given the difficulties and disadvantages associated with currently available bispecific antibodies for T cell mediated cancer immunotherapy, there remains a need for novel, improved formats of such molecules. These drawbacks have now been overcome with the new bispecific antibodies of the invention. The new bispecific antibodies can be easily produced, with an increased yield due to a decreased amount of mispaired side-products, which show less aggregation than bispecific antibody fragments known in the art. In addition, the new bispecific antibodies are stable at increased temperatures compared to bispecific antibody formats known in the art. Using the crossover approach correct chain association can be enforced without the need for the generation of a common light chain. In addition, the new the new bispecific antibodies has a higher molecular weight compared to many conventional bispecific antibody fragments, thus preventing excessive kidney clearance and leading to an improved half-life in vivo. Further, only two plasmids are needed to produce the new bispecific antibodies. The new bispecific antibodies are fully functional and have comparable or improved binding and activity as corresponding conventional bispecific antibodies.
The present invention provides bispecific antigen binding molecules designed for T cell activation and re-direction that combine good efficacy and produceability with low toxicity and favorable pharmacokinetic properties.